Fertilization requires that two dissimilar cells fuse their plasma membranes to create a zygote. This process is poorly understood and there has been no evidence it is well-conserved. We recently discovered C. elegans Izumo-like (CEIZUMO), which is the first case of a homologous membrane protein required for fertilization in both mammals and nematodes. Under specific aim 1, we will identify the location and role of CeIzumo during fertilization in vivo. Our studies will first use immunological approaches to characterize and visualize C. elegans CEIZUMO. This is will determine where CEIZUMO is localized to facilitate cell-cell fusion and reveal other features it might share with other well-characterized C. elegans and mouse fertilization mutants. We will use a transgenic approach to determine which parts of CeIzumo and worm/mouse gene chimeras restore self-fertility to the CeIzumo mutant; we already know that the mouse Ig domain can be swapped into CeIzumo without compromising its function. Under specific aim 2, we will determine if CeIzumo is a C. elegans sperm fusogen in vitro. We will develop an in vitro fertilization system to allow determination of the precise point where CEIZUMO acts. We will also establish whether an extracellular fragment of CEIZUMO can bind to unfertilized eggs. If successful, this will be followed by an RNAi screen for egg surface CEIZUMO receptors. The emerging picture is that worm and mammalian Izumo homologs are orthologous and could define a core cell-cell fusion mechanism that has been conserved during fertilization in animals. Antibodies to Izumo are known to block human sperm-egg fusion, suggesting that this protein is an outstanding contraceptive target. The C. elegans experiments described in this proposal already suggest that a high throughput assay for small molecules that block human Izumo function might be possible. PUBLIC HEALTH RELEVANCE: Fertilization is a process with significant public health implications because ~15% of couples desiring children are infertile and many cases are due to defective sperm. We have discovered that cell-cell-fusion during fertilization in nematodes may be similar to that which occurs in humans. We will exploit the significant experimental advantages offered by nematodes for analyzing this important problem in reproductive biology.